Prion diseases (PrD) are rare and fatal neurodegenerative disorders characterized by the accumulation of insoluble prion protein (PrP) in the brain. Despite compelling evidence for the role of the templated conversion of PrPC into PrPSc in the spread of prions, our current understanding of the primary causes of PrP neurotoxicity is limited. Our long-term goal is to uncover the molecular factors mediating the pathogenesis of PrD. The overall objective of this proposal is to identify the intrinsic and extrinsic triggers of human PrP neurotoxicity in Drosophila. Flies expressing PrP from rodents and sheep display late-onset brain degeneration but lack an easy-to-score phenotype, which has hindered the discovery of the mechanisms mediating PrP toxicity. Since natural variants of PrP seem to affect its disease potential, we postulated that the heightened toxicity of human PrP (HuPrP) underlies the exceptional complexity of human PrD. Our HYPOTHESIS is that HuPrP efficiently folds into highly pathogenic conformations that hijack endogenous cellular components to activate deleterious intracellular signals that kill neurons. We based this hypothesis on extensive literature review and compelling preliminary results demonstrating the robust toxicity of HuPrP in flies. HuPrP-WT kills brain neurons and induces small/glassy eyes in flies. Also, reducing the activity of PERK and its effector ATF4 suppresses the glassy eyes, demonstrating (a) the physiological relevance of HuPrP toxicity in Drosophila and (b) its sensitivity to known mediators of PrP toxicity. The rationale for this proposal is that (1) the toxicity of HuPrP in Drosophila stems from aberrant molecular interactions that can be defined genetically, (2) Drosophila is an ideal model for large modifier screens, and (3) understanding HuPrP toxicity is directly relevant for uncovering the pathogenic cascades in human prion diseases. We will test our central hypothesis with the following Specific Aims: -Working hypothesis I: HuPrP accumulates specific isoforms responsible for its heightened toxicity Aim 1: Identify intrinsic determinants of human PrP toxicity. We will introduce the M129 polymorphism and reverse HuPrP toxicity with the N159D substitution from dog PrP. Then, we will identify the HuPrP-specific neurotoxic isoforms that trigger toxicity and the cellular mechanisms that produce them by biochemical studies. -Working hypothesis II: Unbiased genetic screens will reveal the genetic factors mediating HuPrP toxicity Aim 2: Identify extrinsic factors mediating human PrP toxicity. We will perform a genome-wide genetic screen for modifiers of the robust eye phenotype of HuPrP. Testing 6,600 RNAi and 5,000 overexpression alleles will identify most pathways mediating HuPrP toxicity. The main INNOVATION of this proposal consists on exploiting the robust phenotypes of a new Drosophila model expressing HuPrP to identify both the intrinsic and extrinsic factors mediating HuPrP toxicity. This is SIGNIFICANT because we will tease apart the complex molecular mechanisms triggering HuPrP toxicity, guiding future therapeutic approaches most likely to work in the clinic.